1. Field of the Invention
The present invention relates to an improved method for determining macromolecules such as proteins, and more particularly to a method comprising forming a latent stain image by nucleating polyacrylamide gels containing the macromolecules with a palladium tetramine salt, developing with a physical developer solution, and contacting the developed stain image with a 1-phenyl-2-tetrazoline-5-thione or a salt of a 1-phenyl-1H-tetrazole-5-thiol. 2. Description of Related Art
The detection of macromolecules such as proteins and polypeptides is extremely important in many areas of biology and clinical medicine such as genetic screening and the diagnosis of genetic diseases. The primary laboratory detection and characterization technique for macromolecules is electrophoresis. Electrophoresis is the movement of charged particles in a matrix under the influence of an electric field. Continuing developments in two-dimensional gel electrophoresis have provided the capability of resolving thousands of macromolecules from complex biological mixtures.
U.S. application Ser. No. 495,216, now U.S. Pat. No. 4,552,848, of Yudelson and Johnson discloses a macromolecule electrophoregram visualization procedure of high sensitivity comprising the steps of forming a latent stain image by nucleating polyacrylamide gels containing the macromolecules with a palladium tetramine salt and developing the latent stain image by treating the gel with a specific physical developer solution.
Although this method is useful and has enjoyed widespread acceptance, the resulting developed electrophoregram is somewhat unstable (it is readily oxidized in air by the presence of moisture resulting in image loss). Reducing agents such as sodium hypophosphite have been used as image stabilizers. Such stabilizing solutions, however, require a high concentration of stabilizer compound to effect stabilization. Moreover, these stabilizing solutions fail after the supply of reducing agent is depleted by reaction with oxygen.
Example 2 of U.S. application Ser. No. 495,216 discloses a developer having therein 1-phenyl-2-tetrazoline-5-thione present at a concentration of about 0.0001%. This reference, however, does not teach or suggest the unexpected image stability resulting from the practice of this invention.
Thus, there is a need for an improved method of stabilizing developed electrophoregrams, in using electrophoregram stabilizer solutions having reduced concentration of stabilizer compound and for electrophoregrams having improved image stability.